Feasibility of proposed single-nucleotide polymorphisms as predictive markers for targeted regimens in metastatic colorectal cancer.

BACKGROUND: Surrogate biomarkers for metastatic colorectal cancer (mCRC) are urgently needed to achieve the best outcomes for targeted therapy. METHODS: A clinical association analysis was performed to examine the three single-nucleotide polymorphisms (SNPs) that were previously proposed as markers of chemosensitivity to the cetuximab (124 patients) and bevacizumab regimens (100 patients) in mCRC patients. In addition, biological correlations were examined for the candidate SNPs in terms of their regulatory pathway. RESULTS: For cetuximab regimens, patients homozygous for the wild-type alleles (GG) of LIFR rs3729740 exhibited a 1.9 times greater overall response rate (ORR) and 1.4 months longer progression-free survival (PFS) than those homozygous or heterozygous for the mutant allele (GA and AA; P=0.022 and 0.027, respectively). For bevacizumab regimens, patients homozygous for the minor alleles (TT) of ANXA11 rs1049550 exhibited an ORR twice as high as those homozygous or heterozygous for the ancestral allele (CC and CT; P=0.031). Overall response rate gain was achieved up to 10% in patients with wild-type LIFR rs3729740 patients either with wild-type KRAS or skin toxicity (P=0.001) respectively. Specifically in clones treated with cetuximab and bevacizumab regimens, active p-ERK and MMP-9 expressions were significantly reduced in clones expressing wild-type LIFR rs3729740 (P=0.044) and in those expressing minor-type ANXA11 rs1049550 (P=0.007), respectively. CONCLUSION: LIFR rs3729740 and possibly ANXA11 rs1049550 may be useful as biomarkers for predicting whether mCRC patients are sensitive to relevant target regimens, although further validation in large cohorts is needed.

Applicability of carcinoembryonic antigen-specific monoclonal antibodies to radioimmunoguided surgery for human colorectal carcinoma.

Two carcinoembryonic antigen (CEA)-specific monoclonal antibodies (MAbs), PR1A3 and T84.66, were tested to determine whether they could accurately localize colorectal carcinoma and therefore be applicable in radioimmunoguided surgery (RIGS). Twenty-one tumors by three human colorectal carcinoma cell lines with various levels of CEA expression (KM-12c, C75, and Clone A) were successfully implanted in the intra-abdominal organs of 15 nude mice. The tumors was localized using a portable radioisotope detector (Neoprobe 1000) 48 h after injection of radiolabeled MAbs (10 mCi/mouse) when the precordial counts were <20 per 2 s. Histopathological identification of radiolabeled MAbs were also performed using immunohistochemistry and microautoradiography. Radioactivity counted on a portable radioisotope detector correlated well with that on a gamma counter. The distribution in the blood was significantly greater than in other organs (P < 0.001). Localization indices of the tumor in various organs was from 1.1 to 8.5 in the PR1A3-pretreated mice and 3.0 to 8.6 in the T84.66-pretreated mice. Silver grains and immune staining were distributed in the tumor cells of the PR1A3-pretreated mice, whereas they were in the necrotic debris as well as the tumor cells of the T84.66-pretreated mice. There were significantly more silver grains in the liver in the T84.66-pretreated mice than in the PR1A3-pretreated mice (P = 0.004). The sensitivity and specificity of tumor localization by RIGS were 71.4 and 91.4% in the PR1A3-pretreated mice, whereas they were 60 and 76% in the T84.66-pretreated mice. A study using specific anti-CEA MAbs suggested PR1A3 as an efficient immune probe for RIGS in colorectal carcinoma with a low rate of false-positive detection.

Somatic mutations of the first 14 exons of APC in hamartomatous polyps of the colon.

Although hamartomatous or hyperplastic polyps are rarely accompanied by adenomatous or carcinomatous foci, the role of APC (MIM# 175100) mutations in these polyps is not clear. The neoplastic potential of these polyps was assessed with regard to somatic mutation of the first 14 exons of APC. DNA from 14 hamartomatous polyps (12 patients with juvenile polyp, JP; two patients with Peutz-Jeghers syndrome, PJS) and 27 hyperplastic polyps was used. Exons 1-14 of APC were amplified using verified oligonucleotide primers, and PCR-SSCP analysis was performed. Translation-terminating mutation in exon 15 was also screened using the protein truncation test. All mutations found were transitions or transversions with heterozygous alleles of both wild-type and mutant APC in exons 2, 9, 10, and 11. Four hamartomatous polyps (three from JP and one from PJS) showed seven, new mutations and one common APC variant (codon 486), whereas no hyperplastic polyps demonstrated mutation. APC mutation was not correlated with previous history of colorectal carcinoma or number of polyps. Since all mutations were missense or silent mutations occurred in exons not previously known to have functionally relevant area, their phenotypic implication appeared to be limited.

Carcinoembryonic antigen gene and carcinoembryonic antigen expression in the liver metastasis of colorectal carcinoma.

The presence of the carcinoembryonic antigen (CEA) gene and CEA expression in the liver was tested to identify their possible roles in the liver metastasis of colorectal carcinoma. The CEA gene in the liver was identified by amplifying the CEA-specific N-terminal domain exon with digoxigenin-dUTP labeling in 16 colorectal carcinomas with liver metastases. Next, CEA expression was tested by immunostaining using the anti-CEA monoclonal antibody (T84.66, ATCC). Liver tissues from 13 stomach cancer patients and 12 colorectal cancer patients without liver metastasis were also tested as control groups. Three grades (<25%, 25-50%, and 50%< or =) were given according to the proportion of positive cells. The CEA gene was amplified in the metastatic tumor cells of the liver (2.6 +/- 0.2, mean grade +/- SEM) and their surrounding hepatocytes (1.5 +/- 0.2) in all cases. CEA expression was found in all metastatic tumor cells and 14 cases of the surrounding hepatocytes. Among the control groups, the CEA gene of the hepatocytes was found in 9 cases each of the colorectal and the stomach cancers that did not exhibit CEA expression. The level of serum CEA was related with the numbers and volume of liver metastases, but not with CEA expression in tumor cells and surrounding hepatocytes. The CEA gene in the metastatic tumor cells, not in the hepatocytes, was closely associated with CEA expression in the surrounding hepatocytes (p<0.01). Although the precise mechanism of CEA gene regulation in hepatocytes remains to be proven, the CEA gene in the metastatic tumor of the liver seems to affect CEA expression in the surrounding hepatocytes facilitating liver metastasis in colorectal carcinoma.

Temporal variation of volatile organic compounds and their major emission sources in Seoul, Korea.

This study examines the characteristics of volatile organic compounds (VOCs) and their major emission sources at the Bulgwang site in Seoul, Korea. The annual levels of VOCs (96.2-121.1 ppb C) have shown a decreasing trend from 2004 to 2008. The most abundant component in Seoul was toluene, which accounted for over 23.5 % of the total VOCs on the parts per billion on a carbon basis, and the portions of alkanes with two to six carbons constituted the largest major lumped group, ranging from 40.1 to 48.4 % (45.3 ± 3.7 %) of the total VOCs. Major components of the solvent (toluene, m/p-xylene, o-xylene, and ethylbenzene) showed high in daytime and summer and low in nighttime and winter due mainly to the variation of the ambient temperature. The species mostly emitted from gasoline vapor (i/n-butane, i/n-pentane, n-hexane, and 2-methylpentane) and vehicular exhaust (ethylene, acetylene, and benzene) showed bimodal peaks in the diurnal variation around the commuting hours because of the high traffic volume. For the 14 out of 15 highest concentration species, the weekend effect was only evident on Sundays because of the stepwise implementation of the 5-day work-week system. Principal components analysis (PCA) was applied in order to identify the sources of the 15 highest concentration VOCs and, as a result, three principal components such as gasoline vapor (48.9 %), vehicular exhaust (17.9 %), and evaporation of solvents (9.8 %) were obtained to explain a total of 76.6 % of the data variance. Most influential contributing sources at the sampling site were traffic-related ones although the use of solvent was the dominant emission source based on the official emission inventory.

Structural characterization of rice starch in rice cake modified by Thermus scotoductus 4-alpha-glucanotransferase (TS alpha GTase).

Rice cake was produced with a thermostable 4-alpha-glucanotransferase from Thermus scotoductus (TS alpha GTase). Starch molecular fine structure, texture, and retrogradation for the enzymatically prepared rice cake were investigated and compared to those for control rice cake. The amylose content in TS alpha GTase-treated rice cakes decreased, whereas branched and linear malto-oligosaccharides ranging from maltose to maltoheptaose increased slightly. The average molecular weight of the enzyme-treated rice starch in rice cake decreased as amylopectin macromolecules were cleaved and reorganized into small amylopectin clusters. The number of shorter side chains (degree of polymerization [DP] < 9) increased, whereas the number of longer side chains (DP > 10) decreased through the disproportionation reaction of TS alpha GTase. After 24 h of storage at 4 degrees C, the enzyme-treated samples demonstrated significantly lower melting enthalpy of retrograded starch (0.4 mJ/mg) compared to that of the control (1.4 mJ/mg). The results indicated that TS alpha GTase treatment effectively inhibited starch retrogradation in rice cakes. It is suggested that the reduction of amylose content, the rearrangement of amylopectin, and the production of malto-oligosaccharides caused by TS alpha GTase treatment are responsible for the ineffective molecular reassociation of rice starch in rice cake.

Preclinical application of radioimmunoguided surgery using anti-carcinoembryonic antigen biparatopic antibody in the colon cancer.

Radioimmunoguided surgery (RIGS) has been known as a sophisticated tool to detect micrometastasis intraoperatively. A preclinical model of RIGS was designed to test the possible clinical applicability of the biparatopic antibody in detecting colorectal cancer. The biparatopic antibody was constructed using two anti-carcinoembryonic antigen (CEA)-specific antibodies, T84.66 and PR1A3, reacting against two different epitopes. (125)I-labeled biparatopic antibody was introduced via the principal colonic arteries at the end of operation in 10 operable patients with colon cancer. After 24 h, the radioactivities of the tumors and lymph nodes were counted using the gamma-detecting probe. The radioactivity count was performed ex vivo. The accurate detection in the primary tumors and metastatic lymph nodes were 100 and 88.7% respectively. False-positive detections occurred in 24 of 256 lymph nodes (9.4%), whereas false-negative detections occurred in 5 of them (2%). The most frequent cause of false-positive detection was dissociated radionuclides trapped in the lymphatic tissues. False-negative detections occurred mainly from weak targeting by radiolabeled antibody, probably due to weak expression of tumor CEA. Conclusively, as most detection errors appear to be reduced within 3 days in vivo, the biparatopic antibody can efficiently be applied to the clinical RIGS, thereby facilitating accurate detection and removal of occult cancer foci in colorectal cancer.

Adhesive function of carcinoembryonic antigen in the liver metastasis of KM-12c colon carcinoma cell line.

PURPOSE: Both experimental and clinical results reveal that carcinoembryonic antigen (CEA) seems to mediate some important role in the liver metastasis of colorectal carcinoma cells. The intent of this study was to verify whether adhesive function of CEA might affect liver metastasis in the CEA-expressing colon carcinoma cell line, KM-12c. METHODS: The hepatic binding of [125I]iododeoxyuridine KM-12c cells was measured with or without intravenous CEA pretreatment in four nude mice each. Then, 2 x 10(6) cells of KM-12c were injected into the splenic subcapsule of 57 CEA-pretreated nude mice. KM-12c cells were prepared in phosphate-buffered saline (control, 27 mice) or anti-CEA monoclonal antibody, T84.66 (30 mice). All mice were killed at the end of the eighth week after implant, and tumor nodules were confirmed histologically. RESULTS: Marginal differences of hepatic sequestration were found between the CEA-pretreated mice and the control group. Splenic tumor occurred in 75 percent (18/24) of the control group and in 40 percent (10/25) of the T84.66-pretreated group (P = 0.0107). Forty-two percent (10/24) incurred liver metastasis in the control group, whereas 20 percent (5/25) did so in the T84.66-pretreated group. The number of splenic tumor cells was significantly related to the number and volume of liver metastasis (P = 0.0065). CONCLUSIONS: CEA enhanced liver metastasis predominantly by successful primary tumor implant, whereas primary hepatic entrapment also supported it to some extent in a weakly metastatic colon carcinoma cell line, KM-12c. Tumor cell aggregates seem to be mediated by homophilic binding of CEA molecules, and it is an important mechanism to yield liver metastasis.

Familial juvenile polyposis coli with APC gene mutation.

Familial juvenile polyposis has been known to have malignant potential, but their genetic relation to familial adenomatous polyposis has not been proven yet. Two young brothers with intermittent rectal bleeding revealed multiple juvenile polyposis. Their father had a history of rectal cancer with multiple colonic polyps. Four frequent exons of APC gene mutation were tested from these patients' white blood cells by polyacrylamide gel electrophoresis and sequencing. The 21-yr-old brother had a missense mutation (GAA-->GGA) at codon 1309, whereas the 18-yr-old brother showed a missense mutation (ATA-->GTA) at codon 1304 in exon 15 of APC gene. Three of four first-degree relatives were affected with familial juvenile polyposis, familial juvenile polyposis with adenomatous change, and rectal cancer with multiple polyps. The APC gene mutation of familial juvenile polyposis in this case suggests a genetic relationship with familial adenomatous polyposis.

Mutations at the APC exon 15 in the colorectal neoplastic tissues of serial array.

Although the APC protein is known to participate in cellular proliferation and apoptosis, APC mutations have been thought to play a major role in the early stage of colorectal tumorigenesis. The somatic APC mutation of exon 15 was assessed to determine its impact on various stages of colorectal tumorigenesis. The colorectal neoplastic tissues of serial array studied included sporadic adenomas (group 1, n = 36), adenomas (group 2, n = 33), and carcinomas (group 3, n = 32) in the synchronous adenoma and carcinoma as well as sporadic carcinomas (group 4, n = 36). Aberrant DNA was detected by protein truncation test and confirmed by direct sequencing. The mutation prevalence was 36.1% in group 1, 45.5% in group 2, 59.4% in group 3, and 41.7% in group 4 with no differences among the groups. Among the 18 patients with synchronous adenoma and carcinoma, 9 had mutation in their adenomas and 12 in their carcinomas. The mutation loci and patterns did not differ in adenomas and carcinomas. Mutations in the mutation cluster region (MCR) were much more frequent than in the preceding region of MCR, i.e., 85.7% vs. 14.3%. The mutation prevalence of villous adenomas appeared greater than that of tubular adenoma (3/21 vs. 3/4). Predominant pathogenic mutations at MCR suggest that the APC mutation is implicated in all stages of colorectal tumorigenesis.

hMLH1 and hMSH2 mutations in families with familial clustering of gastric cancer and hereditary non-polyposis colorectal cancer.

The pattern of hMLHI and hMSH2 mutations was assessed to identify the genetic correlation between hereditary gastric and colorectal cancers. Four disease groups and their healthy family members were assembled according to the presentation of gastric cancer: FG, familial clustering of gastric cancer (n = 32); CG, family with one or more colorectal and gastric cancers in first-degree relatives (n = 22); HS, seven HNPCC families corresponding to the Amsterdam criteria (AMS+) and 12 suspected HNPCC families which did not satisfy one of the criteria (AMS-), but no gastric cancer among first- and second-degree relatives (n = 19); and SG, sporadic gastric cancer (n = 33). In the CG group, three were included in AMS + and six in AMS- criteria. Peripheral blood was obtained from them to detect hMLHI and hMLH2 mutations using PCR-SSCP analysis and direct sequencing. The incidence of mutations was 9.4% in the FG group, 54.5% in the CG group, 31.6% in the HS group, and none in the SG group. The incidence, type, and number of the mutation were not different between the CG and HS groups. Thirty-four different mutations included 19 in hMLH1 and 15 in hMSH2. Gastric cancer was the most common extracolonic malignancy in HNPCC and suspected HNPCC families (9/28, 32.1%). The hMLH1 or hMSH2 mutation occurred in seven of 10 families with AMS+, whereas it occurred in four of 18 with AMS- (70% vs. 22.2%, P = .013). Five mutations in the hMLH1 and six mutations in the hMSH2 were exclusively found in families with gastric cancer. All three mutations in the FG group were in hMLHI and there was no mutation in their healthy family members. This study demonstrates that some familial clustering type of gastric cancer appears to be associated with hMLHI mutations thereby indicating a difference from the hereditary gastric cancer studies previously reported. In addition, hMLHI and hMSH2 mutations may impact the gastric cancer carcinogenesis in HNPCC or suspected HNPCC.